Project Summary/Abstract How neural circuits control complex behaviors is a fundamental problem in neuroscience. Optogenetic tools have transformed our ability to identify genetically marked neuronal subpopulations of interest (NSOIs), which serve as ?points-of-entry? to behaviorally relevant circuits. However, a gap remains between the identification of such NSOIs, and the ability to identify and map their downstream target neurons (DTNs); identification of such DTNs is a crucial step in further tracing the circuit. To fill this gap, we will combine optogenetic stimulation of NSOIs with single-cell RNA sequencing (scRNAseq), automated immediate early gene (IEG) mapping, anatomical tracing and electrophysiology in candidate downstream target regions (CDTRs), to identify DTNs of NSOIs in the ventromedial hypothalamus (VMH), which controls aggression, defense and other emotional behaviors. The long-term goal is to elucidate the neural circuits that control decisions between complex social and defensive behaviors. The overall objective of this application is to develop a general approach for identifying and functionally characterizing DTNs of any NSOI of interest. Proof- of-principle for these methods will be obtained by applying them to NSOIs in VMH which have previously been shown to robustly control social and defensive behaviors. Preliminary studies have revealed that optogenetic stimulation of estrogen receptor-1 (Esr1)-expressing neurons in VMHvl in solitary animals can be used to map candidate DTNs throughout the brain, using serial-2-photon tomographic analysis of c-fos expression. This approach will be used to identify candidate DTNs, using scRNAseq and activity-dependent marking systems. The central objective of this proposal is to molecularly identify, and determine the behavioral function, and downstream connectivity, of DTNs to which VMHvlEsr1 neurons (and other behaviorally relevant VMH NSOIs) project. The rationale for this research is that solving this general problem is essential to making forward progress in mapping the circuitry that governs complex behaviors. In Aim 1, we will develop an approach for unbiased brain-wide mapping of DTNs activated by optogenetic stimulation of a genetically defined NSOI. In Aim 2, we will develop and apply a new method, opto-Act-seq, to transcriptomically identify activated DTNs in CDTRs. In Aim 3, we will identify projection DTNs (DTN-PNs) that are monosynaptic targets of the NSOIs, using the novel methods ?opto-TRACM? and ?CRACM/TRACM-guided Patch-seq?. In Aim IV, we will determine the functional role of DTN-PNs in social behaviors using ?multiplex opto-TRACM.? The contribution will be to develop a new approach to identify functionally and behaviorally relevant downstream targets of NSOIs in a systematic, brain-wide manner. This contribution is significant because it allows unbiased, global mapping of downstream circuits activated by a given NSOI. The approach is innovative, because it will develop novel tools, and apply them to a behaviorally important circuit. The work proposed in this application will, therefore, both advance knowledge in this specific field, and will also provide tools for investigators study other circuits.